Hydrazone Cross-linking Research Articles

Self-adjuvanted L-arginine-modified dextran-based nanogels for sustained local antigenic protein delivery to antigen-presenting cells and enhanced cellular and humoral immune responses.

In the development of cancer vaccines, antigens are delivered to elicit potent and specific T-cell responses to eradicate tumour cells. Nonetheless, successful vaccines are often hampered by the poor immunogenicity of tumour antigens, rapid clearance by the innate immunity, and limited cross-presentation on MHC-I to activate CD8+ T-cells arm. To address these issues, we developed dextran-based nanogels to promote antigen uptake, storage, and cross-presentation on MHC-I, while directing immunogenic maturation of the antigen-presenting cells (APCs). To promote the nanocarriers interaction with cells, we modified DX with L-arginine (Arg), whose immunomodulatory activities have been well documented. The ArgDX nanogel performance was compared with the nanogel modified with L-histidine (His) and L-glutamate (Glut). Moreover, we introduced pH-sensitive hydrazone crosslinking during the nanogel formation for the conjugation and controlled release of antigen ovalbumin (OVA). The OVA-laden nanogels have an average size of 325 nm. We demonstrated that the nanogels could rapidly release cargoes upon a pH change from 7 to 5 within 8 days, indicating the controlled release of antigens in the acidic cellular compartments upon internalization. Our results revealed that the ArgDX nanogel could promote greater antigen uptake and storage in DCs in vitro and promoted a stronger immunogenic maturation of DCs and M1 polarization of the macrophages. The OVA signals were co-localized with lysosomal compartments up till 96 hours post-treatment and washing, suggesting the nanogels could facilitate prolonged antigen storage and supply from endo-lysosomal compartments. Furthermore, all the tested nanogel formulations retained antigens at the skin injection sites until day 21. Such delayed clearance could be due to the formation of micron-sized aggregates of OVA-laden nanogels, extending the interactions with the resident DCs. Amongst the amino acid modifications, ArgDX nanogels promoted the highest level of lymph node homing signal CCR7 on DCs. The nanogels also showed higher antigen presentation on both MHC-I and II than DX in vitro. In the in vivo immune studies, ArgDX nanogels were more superior in inducing cellular and humoral immunity than the other treatment groups on day 21 post-treatment. These results suggested that ArgDX nanogel is a promising self-adjuvanted nanocarrier for vaccine delivery.

Injectable On-Demand Pulsatile Drug Delivery Hydrogels Using Alternating Magnetic Field-Triggered Polymer Glass Transitions.

Remote-controlled pulsatile or staged release has significant potential in a wide range of therapeutic treatments. However, most current approaches are hindered by the low resolution between the on- and off-states of drug release and the need for surgical implantation of larger controlled-release devices. Herein, we describe a method that addresses these limitations by combining injectable hydrogels, superparamagnetic iron oxide nanoparticles (SPIONs) that heat when exposed to an alternating magnetic field (AMF), and polymeric nanoparticles with a glass transition temperature (Tg) just above physiological temperature. Miniemulsion polymerization was used to fabricate poly(methyl methacrylate-co-butyl methacrylate) (p(MMA-co-BMA)) nanoparticles loaded with a model hydrophobic drug and tuned to have a Tg value just above physiological temperature (∼43 °C). Co-encapsulation of these drug-loaded nanoparticles with SPIONs inside a carbohydrate-based injectable hydrogel matrix (formed by rapid hydrazone cross-linking chemistry) enables injection and immobilization of the nanoparticles at the target site. Temperature cycling facilitated a 2.5:1 to 6:1 on/off rhodamine release ratio when the nanocomposites were switched between 37 and 45 °C; release was similarly enhanced by exposing the nanocomposite hydrogel to an AMF to drive heating, with enhanced release upon pulsing observed even 1 week after injection. Coupled with the apparent cytocompatibility of all of the nanocomposite components, these injectable nanocomposite hydrogels are promising as minimally invasive but remotely actuated release delivery vehicles capable of complex release kinetics with high on-off resolution.

Hyaluronic acid based next generation bioink for 3D bioprinting of human stem cell derived corneal stromal model with innervation

Corneal transplantation remains gold standard for the treatment of severe cornea diseases, however, scarcity of donor cornea is a serious bottleneck. 3D bioprinting holds tremendous potential for cornea tissue engineering (TE). One of the key technological challenges is to design bioink compositions with ideal printability and cytocompatibility. Photo-crosslinking and ionic crosslinking are often used for the stabilization of 3D bioprinted structures, which can possess limitations on biological functionality of the printed cells. Here, we developed a hyaluronic acid-based dopamine containing bioink using hydrazone crosslinking chemistry for the 3D bioprinting of corneal equivalents. First, the shear thinning property, viscosity, and mechanical stability of the bioink were optimized before extrusion-based 3D bioprinting for the shape fidelity and self-healing property characterizations. Subsequently, human adipose stem cells (hASCs) and hASC-derived corneal stromal keratocytes were used for bioprinting corneal stroma structures and their cell viability, proliferation, microstructure and expression of key proteins (lumican, vimentin, connexin 43, α-smooth muscle actin) were evaluated. Moreover, 3D bioprinted stromal structures were implanted into ex vivo porcine cornea to explore tissue integration. Finally, human pluripotent stem cell derived neurons (hPSC-neurons), were 3D bioprinted to the periphery of the corneal structures to analyze innervation. The bioink showed excellent shear thinning property, viscosity, printability, shape fidelity and self-healing properties with high cytocompatibility. Cells in the printed structures displayed good tissue formation and 3D bioprinted cornea structures demonstrated excellent ex vivo integration to host tissue as well as in vitro innervation. The developed bioink and the printed cornea stromal equivalents hold great potential for cornea TE applications.

Dynamically Crosslinked Poly(ethylene-glycol) Hydrogels Reveal a Critical Role of Viscoelasticity in Modulating Glioblastoma Fates and Drug Responses in 3D.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain tumor in adults. Hydrogels have been employed as 3D in vitro culture models to elucidate how matrix cues such as stiffness and degradation drive GBM progression and drug responses. Recently, viscoelasticity has been identified as an important niche cue in regulating stem cell differentiation and morphogenesis in 3D. Brain is a viscoelastic tissue, yet how viscoelasticity modulates GBM fate and drug response remains largely unknown. Using dynamic hydrazone crosslinking chemistry, a poly(ethylene-glycol)-based hydrogel system with brain-mimicking stiffness and tunable stress relaxation is reported to interrogate the role of viscoelasticity on GBM fates in 3D. The hydrogel design allows tuning stress relaxation without changing stiffness, biochemical ligand density, or diffusion. The results reveal that increasing stress relaxation promotes invasive GBM behavior, such as cell spreading, migration, and GBM stem-like cell marker expression. Furthermore, increasing stress relaxation enhances GBM proliferation and drug sensitivity. Stress-relaxation induced changes on GBM fates and drug response are found to be mediated through the cytoskeleton and transient receptor potential vanilloid-type 4. These results highlight the importance of incorporating viscoelasticity into 3D in vitro GBM models and provide novel insights into how viscoelasticity modulates GBM cell fates.

Highly Efficient and Robust Monolith of a Metal–Organic Framework Hybrid Aerogel for the Removal of Antimony from Water

Antimony contamination is of increasing concern across various environmental media. Removal of antimony from water is a pressing issue, and an associated challenge in the operational treatment of wastewater is developing material of high Sb absorbance efficiency together with facile applicability. This work details, for the first time, a 3D porous hybrid aerogel comprising metal–organic frameworks (MOFs) embedded in the cellulose nanocrystal matrix using a bottom-up approach based on hydrazone cross-linking of hydrazide and aldehyde, specifically designed for removal of Sb(V) from contaminated water. The hybrid aerogel was fabricated, and the resulting product presents a monolithic architecture with a diameter of 2 cm and a super low density of 7.96 mg cm–3. The hybrid aerogel has fast adsorption kinetics, a broad working pH range, strong anti-interference capability, and high selectivity. In particular, the material has an ultrahigh adsorption capacity (575 mg g–1) for Sb(V) that is far superior to other adsorbents. Site energy thermodynamic and mass transfer modeling results demonstrated that the cross-linked celluloses greatly disperse and protect the embedded MOFs so that abundant active Bi–O sites strongly adsorb Sb(V) by forming Bi–O–Sb bonds. Almost complete (97%) removal of Sb(V) from 5 mL of spiked tap water (50 μg L–1) was achieved in 2 min by simple extrusion through a syringe device assembled with blocks of the hybrid aerogel and is well below the maximum contaminant level for Sb(V) of 6 μg L–1 in drinking water. This work paves the way for shaping powder adsorbents into convenient and tailorable free-standing monoliths, and with these features the hybrid aerogel is a promising candidate for practical treatment of Sb(V)-contaminated water.

Hyaluronic acid-based biphasic scaffold with layer-specific induction capacity for osteochondral defect regeneration

The repair of cartilage and subchondral bone defects, especially the former, remains a challenge in clinical practice. An increasing number of studies have shown that the use of biomaterials has great advantages in regulating tissue repair, with lower cost and risk than the use of cytokines or cells. However, great challenges remain because of the marked differences between cartilage and subchondral bone. Here, biphasic osteochondral scaffolds with layer-specific differentiation were designed and developed. Hyaluronic acid (HA) was used as the matrix in the upper and lower layers, and differences in physicochemical properties and biological properties between the two layers were realized by different chemical crosslinking methods. The cartilage regeneration layer exhibited stress relaxation from the HA hydrazone crosslinking dynamics. Additionally, a porous HA hydrogel with dominant elasticity was prepared for subchondral bone repair. Importantly, a 3D-printed scaffold bracket could keep close integration of two layers and provided mechanical support, aiding the long-term tissue regeneration process. The in vitro and in vivo results indicated that the HA-base bilayer scaffold consisting of a dynamic HA layer and a porous HA layer realized the simultaneous integrated regeneration of articular cartilage and subchondral bone. Such scaffolds represent potential new materials for osteochondral regeneration.

Interpenetrating gallol functionalized tissue adhesive hyaluronic acid hydrogel polarizes macrophages to an immunosuppressive phenotype

Innovative scaffold designs that modulate the local inflammatory microenvironment through favorable macrophage polarization and suppressing oxidative stress are needed for successful clinical translation of regenerative cell therapies and graft integration. We herein report derivation of a hydrazone-crosslinked gallol functionalized hyaluronic acid (HA-GA)-based hydrogel that displayed outstanding viscoelastic properties and immunomodulatory characteristics. Grafting of 6% gallol (GA) to a HA-backbone formed an interpenetrative network by promoting an additional crosslink between the gallol groups in addition to hydrazone crosslinking. This significantly enhanced the mechanical stability and displayed shear-thinning/self-healing characteristics, facilitated tissue adhesive properties to porcine tissue and also displayed radical scavenging properties, protecting encapsulated fibroblasts from peroxide challenge. The THP-1 human macrophage cell line or primary bone-marrow-derived murine macrophages cultured within HA-GA gels displayed selective polarization to a predominantly anti-inflammatory phenotype by upregulating IL4ra, IL-10, TGF-β, and TGF-βR1 expression when compared with HA-HA gels. Conversely, culturing of pro-inflammatory activated primary murine macrophages in HA-GA gels resulted in a significant reduction of pro-inflammatory TNF-α, IL-1β, SOCS3 and IL-6 marker expression, and upregulated expression of anti-inflammatory cytokines including TGF-β. Finally, when the gels were implanted subcutaneously into healthy mice, we observed infiltration of pro-inflammatory myeloid cells in HA-HA gels, while immunosuppressive phenotypes were observed within the HA-GA gels. Taken together these data suggest that HA-GA gels are an ideal injectable scaffold for viable immunotherapeutic interventions. Statement of significanceHost immune response against the implanted scaffolds that are designed to deliver stem cells or therapeutic proteins in vivo significantly limits the functional outcome. For this reason, we have designed immunomodulatory injectable scaffolds that can favorably polarize the recruited macrophages and impart antioxidant properties to suppress oxidative stress. Specifically, we have tailored a hyaluronic acid-based extracellular matrix mimetic injectable scaffold that is grafted with immunomodulatory gallol moiety. Gallol functionalization of hydrogel not only enhanced the mechanical properties of the scaffold by forming an interpenetrating network but also induced antioxidant properties, tissue adhesive properties, and polarized primary murine macrophages to immunosuppressive phenotype. We believe such immunoresponsive implants will pave the way for developing the next-generation of biomaterials for regenerative medicine applications.

Nanostructured degradable macroporous hydrogel scaffolds with controllable internal morphologies via reactive electrospinning

Creating micro/nanostructured hydrogels with tunable morphologies under cell-friendly processing conditions would enable rational engineering of hydrogel scaffolds for targeted biomedical applications. Herein, an all-aqueous single-step reactive electrospinning method is applied to prepare hydrogel networks with controlled morphologies on both the nanoscale and the microscale. Hydrazide and aldehyde-functionalized poly(oligo ethylene glycol methacrylate) (POEGMA) are co-spun from a double barrel syringe together with poly(ethylene oxide) (PEO) as an electrospinning aid. By varying the concentrations and molecular weights of PEO and/or POEGMA, various morphologies from pure fibers to beaded fibers to bead network morphologies with tunable bead sizes can be fabricated, all of which remain monolithically stable in water due to the dynamic covalent crosslinks formed within the gel structure. The rates and magnitudes of swelling, degradation, and mechanics of the resulting scaffolds can be tuned by independently controlling gel morphologies on the nanoscale (i.e. crosslink density within the gel) and the microscale (i.e. the network structure formed), with an atypical independence of swelling relative to the mechanics and degradation rate observed. Furthermore, the internal morphology of the networks is demonstrated to systematically alter both the cell responses within the scaffolds and the rate of protein release from the scaffolds, with small fibers showing optimal cell proliferation, bead networks exhibiting the slowest protein release kinetics and very high maintained cell viabilities post-electrospinning, and beaded fibers showing intermediate properties. Statement of SignificanceControlling the internal structure of hydrogels is critical to successfully applying hydrogels in biomedical applications such as tissue engineering or cell/drug delivery. However, current techniques to fabricate hydrogel scaffolds typically require additives or gelation processes that are poorly compatible with cells and/or require multi-step processes. In this paper, we describe the fabrication of hydrogel scaffolds with tunable feature sizes (from nanometer to micrometer scale) and structures (from all fibers to bead/fiber mixtures to a new “bead network” morphology) using a reactive electrospinning strategy leveraging dynamic hydrazone crosslinking. We show single-step cell/protein loading and systematic control over cell proliferation and protein release kinetics by systematically manipulating the scaffold morphologies and feature sizes, allowing facile customization of scaffold properties for targeted applications.

Impact of Hydrogel Cross-Linking Chemistry on the in Vitro and in Vivo Bioactivity of Recombinant Human Bone Morphogenetic Protein-2.

Designing strategies to deliver functional proteins at physiologically relevant concentrations using chemically cross-linked biocompatible hydrogels is a major field of research. However, the impact of cross-linking chemistry on the encapsulated protein bioactivity is rarely studied. Here we examine the two well-known cross-linking reactions namely; hydrazone cross-linking chemistry and thiol-Michael addition reaction to form hyaluronic acid (HA) hydrogels. As a therapeutic protein, we employed recombinant human bone morphogenetic protein-2 (rhBMP-2) for this study. Incubation of rhBMP-2 with HA functionalized with a thiol diminished phosphorylation of Smad 1/5/8, a signal transducer for osteogenic differntiation, whereas an aldehyde functionalized HA had no effect. This indicates that thiol functionalized polymers indeed has an impact on protein function. To validate this result in an in vivo setting we performed BMP-2 induced bone formation in a rat ectopic model. These experiments revealed that the hydrazone-cross-linked HA-hydrogel induced significantly higher bone formation (18.90 ± 4.25 mm3) as compared to the HA-thiol-Michael hydrogels (1.25 ± 0.52 mm3) after 8 weeks as determined by micro-computed tomography. The histological examination of the neo-bone indicated that hydrazone-hydrogels promoted a better quality of bone formation with improved mineralization and collagen formation as compared to the thiol-Michael hydrogels. We believe such a direct comparison of two cross-linking chemistries will provide new insight for developing biomaterials for protein delivery for in vivo applications.

Post-synthesis modification of thermo-responsive hydrogels: Hydrazone crosslinking of α-oxoaldehyde obtained from NIPAm-based polymers

Currently, the chemistry of alpha-oxoaldehydes (α-oxo-ALD) is a versatile and useful tool in the field of bioconjugation of peptides and proteins. The combination of smart materials with biomolecules also represents an important opportunity for the development of smart functional materials in a wide range of applications. However, the development of thermosensitive polymeric materials with α-oxo-ALD has not yet been reported. For this reason, the incorporation of these highly reactive functional groups in a thermo-responsive polymer by a successful protocol is reported in this work. Poly-N-isopropylacrylamide (p-NIPAm) functionalized with α-oxo-ALD (p-NIPAm-α-oxo-ALD) was obtained from precursor hydrogels (HGs) based on NIPAm crosslinked with (+)-N,N′-diallyltartramide (DAT). This crosslinker presents vicinal hydroxyl groups which can be cleaved with periodate to yield nanoparticles (10–100 nm range) with pendant α-oxo-ALD groups. Furthermore, the p-NIPAm-α-oxo-ALD product obtained was used to synthesize novel thermo-responsive hydrogels by its reaction with adipic acid dihydrazide (AADH). Then, the formation of hydrazone bonds at different pHs and the effect of thermal transition of p-NIPAm on the gelation were studied. The developed protocol may be fully investigated as a platform to obtain materials for specific applications.

Characterization of the microstructure of hydrazone crosslinked polysaccharide-based hydrogels through rheological and diffusion studies

Microstructure plays an essential role in the control of hydrogel properties. It is also an important factor when cells or drugs are encapsulated inside the hydrogel. In this work, the microstructures of hydrazone crosslinked hyaluronan-, alginate- and gellan gum-based hydrogels were evaluated thoroughly for the first time by using rheology- and diffusion (fluorescence recovery after photobleaching, FRAP)-based methods. The effect of gel parameters on the viscoelastic and diffusion properties of hydrogels, and further on their structural parameters (mesh size, average molecular weight of the polymer chain between neighboring crosslinks, crosslinking density) are shown. Results further show that diffusivity decreased when larger dextran sizes were used, which were equivalent to the mesh sizes of hydrogels (15 nm to 47 nm) evaluated by the rheological method. This mesh size range allows the transportation of smaller molecules, but also peptides and most of the proteins. A correlation between the storage modulus and the structural parameters was also shown. Overall, hydrazone crosslinking offers an easy way to produce polysaccharide-based hydrogels with variable microstructures by altering the gel parameters.

Dynamic Hyaluronan Hydrogels with Temporally Modulated High Injectability and Stability Using a Biocompatible Catalyst

Injectable and biocompatible hydrogels have become increasingly important for cell transplantation to provide mechanical protection of cells during injection and a stable scaffold for cell adhesion post-injection. Injectable hydrogels need to be easily pushed through a syringe needle and quickly recover to a gel state, thus generally requiring noncovalent or dynamic cross-linking. However, a dilemma exists in the design of dynamic hydrogels: hydrogels with fast exchange of cross-links are easier to eject using less force, but lack long-term stability; in contrast, slow exchange of cross-links improves stability, but compromises injectability and thus the ability to protect cells under flow. A new concept to resolve this dilemma using a biocompatible catalyst to modulate the dynamic properties of hydrogels at different time points of application to have both high injectability and high stability is presented. Hyaluronic acid based hydrogels are formed through dynamic covalent hydrazone cross-linking in the presence of a biocompatible benzimidazole-based catalyst. The catalyst accelerates the formation and exchange of hydrazone bonds, enhancing injectability, but rapidly diffuses away from the hydrogel after injection to retard the exchange and improve the long-term stability for cell culture.

Dynamically Cross-Linked Self-Assembled Thermoresponsive Microgels with Homogeneous Internal Structures.

The internal morphology of temperature-responsive degradable poly(N-isopropylacrylamide) (PNIPAM) microgels formed via an aqueous self-assembly process based on hydrazide and aldehyde-functionalized PNIPAM oligomers is investigated. A combination of surface force measurements, small angle neutron scattering (SANS), and ultrasmall angle neutron scattering (USANS) was used to demonstrate that the self-assembled microgels have a homogeneously cross-linked internal structure. This result is surprising given the sequential addition process used to fabricate the microgels, which was expected to result in a densely cross-linked shell-diffuse core structure. The homogeneous internal structure identified is also significantly different than conventional microgels prepared via precipitation polymerization, which typically exhibit a diffuse shell-dense core structure. The homogeneous structure is hypothesized to result from the dynamic nature of the hydrazone cross-linking chemistry used to couple with the assembly conditions chosen that promote polymer interdiffusion. The lack of an internal cross-linking gradient within these degradable and monodisperse microgels is expected to facilitate more consistent drug release over time, improved optical properties, and other potential application benefits.

Hydrazone crosslinked hyaluronan-based hydrogels for therapeutic delivery of adipose stem cells to treat corneal defects

Corneal blindness is a worldwide problem, plagued by insufficient amount of high-quality donor tissue. Cell therapy using human adipose stem cells (hASCs) has risen as an alternative to regenerate damaged corneal stromal tissue, the main structural and refractive layer of the cornea. Herein we propose a method to deliver hASCs into corneal defects in hyaluronan (HA)-based hydrogels, which form rapidly in situ by hydrazone crosslinking. We fabricated two different HA-based hydrazone-crosslinked hydrogels (HALD1-HACDH and HALD2-HAADH), and characterized their swelling, degradation, mechanical, rheological and optical properties and their ability to support hASC survival. To promote hASC attachment and survival, we incorporated collagen I (col I) to the more stable HALD1-HACDH hydrogel, since the HALD2-HAADH hydrogel suffered swift degradation in culture conditions. We then used an organ culture model with excised porcine corneas to study the delivery of hASCs in these three hydrogels for stromal defect repair. Although all hydrogels showed good hASC survival directly after encapsulation, only the collagen-containing HALD1-HACDH-col I hydrogel showed cells with elongated morphology, and significantly higher cell metabolic activity than the HALD1-HACDH gel. The addition of col I also increased the stiffness and reduced the swelling ratio of the resulting hydrogel. Most importantly, the corneal organ culture model demonstrated these hydrogels as clinically feasible cell delivery vehicles to corneal defects, allowing efficient hASC integration to the corneal stroma and overgrowth of corneal epithelial cells.

Self-crosslinking and injectable chondroitin sulfate/pullulan hydrogel for cartilage tissue engineering

Injectable hydrogels derived from natural polysaccharides have great application prospects in cartilage tissue engineering (CTE). In this study, chondroitin sulfate was functionalized with adipic dihydrazide (CS-ADH), and pullulan was oxidized (oxPL). We fabricated a self-crosslink, biodegradable, in situ forming, injectable hydrogel by covalent hydrazone crosslinking of polysaccharides, without using extra cross-linking agents and releasing harmful byproducts under physiological conditions. The gelation time, equilibrium swelling, degradation behavior, network morphology and mechanical properties of CS-ADH/oxPL hydrogels were well manipulated by varying the weight ratio and concentration of CS-ADH and oxPL. In addition, encapsulation of rabbit articular chondrocytes in CS-ADH/oxPL hydrogels was observed after 14 days culture. Live/Dead staining assay demonstrated that the hydrogel system possessed good cytocompatibility. Sulfated glycosaminoglycan (sGAG) contents indicated that the hydrogel system favored cartilaginous extracellular matrix (ECM) deposition. Furthermore, the hydrogel system could maintain the chondrocyte phenotype and enhance chondrogenesis, which was evaluated in terms of their protein and gene expression. These features support that CS-ADH/oxPL hydrogels have a potential as an injectable cell delivery carrier scaffold in CTE.

Light-activatable prodrugs based on hyaluronic acid biomaterials

Photosensitive in situ cross-linked hyaluronan (HA) hydrogels are prepared by modular chemoselective assembly from the biopolymer precursors and novel heterobifunctional linkers with middle photo-labile ortho-nitrobenzyl group and orthogonally reactive terminals. Starting from the thiol-modified HA and a linker with activated disulfide and hydrazide terminals, a photo-degradable HA hydrogel was prepared by the hydrazone cross-linking reaction. Moreover, a light-triggered drug-releasing hydrogel prodrug was constructed by an orthogonal conjugation of dopamine (DA) via a photo-labile linker to HA dually modified with thiol and hydrazide groups (hy-HA-SH) and a subsequent cross-linking with aldehyde-derivatized HA (HA-al). On-demand release of DA from the hydrogel was achieved upon exposure of the hydrogel to UV light whereas 11-fold less release of the drug was observed in the absence of light. The mechanical properties of the hydrogels, photodegradation kinetics, photorelease of the model drugs were studied by rheology, spectrophotometry, chromatography, and mass spectrometry. For the first time, integration of photolabile components into an actual polysaccharide of extracellular matrix was implemented for the light-controlled release of drug molecules.

Hydrazone cross-linked micelles based on redox degradable block copolymer for enhanced stability and controlled drug release

In this work, an amphiphilic copolymer, PCL-SS-P(PEGMA-co-MAEBA), which contained a disulfide joint in backbone was designed and synthesized. The subsequent micelles that self-assembled from the copolymers were cross-linked by hydrazone, resulting in novel stimuli-responsive degradable micelles with a reversible cross-linked shell. By way of the hydrazone cross-linking of the micellar shell, SCMs owned a good stability against the extensive dilution by water or organic solvent. Doxorubicin (DOX) was used as the model drug for studying the in vitro release profiles of the SCMs. In normal physiological conditions at pH7.4, a quite slow speed was observed with DOX release (only 23% after 72h); when conditions were changed to pH5.0, the SCMs successfully de-crosslinked, DOX release was accelerated (62%). Moreover, drug release was further promoted and reached 87% when 10mM GSH was present, which was primarily due to the breakage of the disulfide joint. The intracellular uptake assay proved that DOX from DOX-loaded SCMs could be efficiently delivered into HepG2 cells after 12h incubation. MTT assays confirmed that DOX-loaded SCMs owned a high cytotoxicity against HepG2 cells. These redox-responsive, degradable SCMs could be a potential candidate for efficient insoluble anticancer drug delivery and therapy.

The production of injectable hydrazone crosslinked gellan gum-hyaluronan-hydrogels with tunable mechanical and physical properties

Gellan gum (GG) has been proposed for use in tissue engineering (TE) due to its structural and functional similarities with alginate. The most traditional crosslinking methods of GG, ionical and photocrosslinking, have downsides such as loss of stability or phototoxicity, which can limit their use in certain applications. In this study, an alternative hydrazone crosslinking method is introduced. Hydrazone crosslinking is a simple method that produces no toxic reagents or side-products. The method enables the fabrication of injectable hydrogels. GG was combined with hyaluronan (HA) to improve some properties such as cell attachment. The mechanical and physical properties of GG-HA hydrogels were controlled by changing the molecular weight, the degree of modification, and the ratio of polymer components. GG-HA hydrogels showed ionic nature of deswelling in the presence of cations enabling the control of physical properties in different solution environments. Due to the non-linear elastic behavior of hydrogels and tissues, the stiffness as a function of strain was represented instead of solely giving the second-order elastic constants. The stiffness of GG-HA hydrogels was similar to that of soft tissues at small strains.

Self-crosslinking and injectable hyaluronic acid/RGD-functionalized pectin hydrogel for cartilage tissue engineering

In the present study, we developed a biomimetic injectable hydrogel system based on hyaluronic acid-adipic dihydrazide and the oligopeptide G4RGDS-grafted oxidized pectin, in which their hydrazide and aldehyde-derivatives enable covalent hydrazone crosslinking of polysaccharides. The hydrazone crosslinking strategy is simple, while circumventing toxicity, making this injectable system feasible, minimally invasive and easily translatable for regenerative purposes. By varying their weight ratios, the physicochemical properties of the mechanically stable hydrogel system were easily adjustable. Additionally, the preliminary studies demonstrated that chondrocyte behavior was dependent on HA/pectin composition and the presence of integrin binding moieties. Specifically, the incorporation of a certain amount of G4RGDS oligopeptide into HA/pectin-based hydrogels could serve as a biologically active microenvironment that supported chondrocyte phenotype and facilitated chondrogenesis. Furthermore, the hydrogel system exhibited acceptable tissue compatibility by using a mouse subcutaneous implantation model. Overall, the novel injectable multicomponent hydrogel presented here is expected to be useful biomaterial scaffold for cartilage tissue regeneration.

A Synthetic Toolbox for the In Situ Formation of Functionalized Homo- and Heteropolysaccharide-Based Hydrogel Libraries.

A synthetic toolbox for the introduction of aldehydo and hydrazido groups into the polysaccharides hyaluronic acid, alginate, dextran, pullulan, glycogen, and carboxymethyl cellulose and their use for hydrogel formation is reported. Upon mixing differently functionalized polysaccharides derived from the same natural precursor, hydrazone cross-linking takes place, which results in formation of a hydrogel composed of one type of polysaccharide backbone. Likewise, hydrogels based on two different polysaccharide strands can be formed after mixing the corresponding aldehydo- and hydrazido-modified polysaccharides. A second line of these studies paves the way to introduce a biomedically relevant ligand, namely, the adhesion factor cyclic RGD pentapeptide, by using an orthogonal click reaction. This set of modified polysaccharides served to create a library of hydrogels that differ in the combination of polysaccharide strands and the degree of cross-linking. The different hydrogels were evaluated with respect to their rheological properties, their ability to absorb water, and their cytotoxicity towards human fibroblast cell cultures. None of the hydrogels studied were cytotoxic, and, hence, they are in principal biocompatible for applications in tissue engineering.